Use of hdac and/or dnmt inhibitors for treatment of ischemic injury

ABSTRACT

The present invention provides methods of ameliorating or reducing the extent of ischemic injury, reperfusion injury, and myocardial infarction, by administering an inhibitor of histone deacetylase enzyme (HDAC) or an inhibitor of DNA methyltransferase enzyme (DNMT).

FIELD OF THE INVENTION

The present invention provides methods of ameliorating or reducing theextent of ischemic injury, reperfusion injury, and myocardialinfarction, by administering an inhibitor of histone deacetylase enzyme(HDAC) or an inhibitor of DNA methyltransferase enzyme (DNMT).

BACKGROUND OF THE INVENTION

Ischemic injury to body tissues such as heart, brain, and lung isresponsible for a significant amount of mortality and morbidity in bothdeveloped and developing countries. Ischemic injury can be induced byevents such as myocardial infarction, stroke, and cardiac surgery.

For example, myocardial ischemia can be associated with myocardialinfarction. Limitation of infarct size is a major goal of therapy foracute coronary syndromes. Strategies of limiting infarct size havefocused on maintaining patency of infarct-related vessels, or providingglucose and insulin to provide necessary metabolic substrates toischemic tissues. But significant improvement is required in thesemethods in order to successfully treat events such as myocardialinfarction in a large percentage of cases.

SUMMARY OF THE INVENTION

The present invention provides methods of ameliorating or reducing theextent of ischemic injury, reperfusion injury, and myocardialinfarction, by administering an inhibitor of histone deacetylase enzyme(HDAC) or an inhibitor of DNA methyltransferase enzyme (DNMT).

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forameliorating an ischemic injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing an extent of an ischemic injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing a size of a myocardial infarction.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing a morbidity of a myocardial infarction.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forameliorating an ischemic injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing an extent of an ischemic injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing a size of a myocardial infarction.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing a morbidity of a myocardial infarction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Effect of ischemia and HDAC inhibitors on histone acetylation(C—non ischemic control; D—ischemic, treated with DMSO; T1—treated withTSA, 1 hour before ischemia).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of ameliorating or reducing theextent of ischemic injury, reperfusion injury, and myocardialinfarction, by administering an inhibitor of histone deacetylase enzyme(HDAC) or an inhibitor of DNA methyltransferase enzyme (DNMT).

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forameliorating an ischemic injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition fortreating an ischemic injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing an extent of an ischemic injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forameliorating an ischemia-reperfusion injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing an extent of a reperfusion injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forameliorating a reperfusion injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing an extent of an ischemia-reperfusion injury.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing a size of a myocardial infarction.

In one embodiment, the present invention provides a use of an inhibitorof an HDAC for the preparation of a pharmaceutical composition forreducing a morbidity of a myocardial infarction.

As provided herein, the findings in Example 2 and 3 demonstrate thatHDAC inhibition reduces myocardial infarct volume in a clinicallyrelevant model of myocardial infarction. The findings of Example 4 showthat HDAC inhibition prevents reduction of histone deacetylation inresponse to ischemia, further confirming the utility of HDAC inhibitorsin reducing ischemic damage. Thus, HDAC inhibitors have multipleapplications in both treating and reducing the incidence of tissueinjury associated with ischemia and subsequent reperfusion, reducingmorbidity and mortality of myocardial infarction, etc.

In one embodiment, the present invention provides a method ofameliorating an ischemic injury in a tissue of a subject, comprisingadministering to the subject an inhibitor of an HDAC, therebyameliorating an ischemic injury in a tissue of a subject.

In one embodiment, the present invention provides a method of treatingan ischemic injury in a tissue of a subject, comprising administering tothe subject an inhibitor of an HDAC, thereby treating an ischemic injuryin a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of an ischemic injury in a subject, comprisingadministering to the subject an inhibitor of an HDAC, thereby reducingan extent of an ischemic injury in a subject.

In another embodiment, the present invention provides a method ofreducing an incidence of an ischemic injury in a subject, comprisingadministering to the subject an inhibitor of an HDAC, thereby reducingan incidence of an ischemic injury in a subject.

“Ischemic injury” refers, in one embodiment, to injury to a tissueresulting from oxygen deprivation. In another embodiment, the termrefers to injury resulting from reperfusion subsequent to oxygendeprivation. In another embodiment, the term refers to injury resultingfrom oxygen deprivation combined with subsequent reperfusion. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the present invention provides a method ofameliorating an ischemia-reperfusion injury in a tissue of a subject,comprising administering to the subject an inhibitor of an HDAC, therebyameliorating an ischemia-reperfusion injury in a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of an ischemia-reperfusion injury in a subject,comprising administering to the subject an inhibitor of an HDAC, therebyreducing an extent of an ischemia-reperfusion injury in a subject.

In one embodiment, the present invention provides a method ofameliorating a reperfusion injury in a tissue of a subject, comprisingadministering to the subject an inhibitor of an HDAC, therebyameliorating a reperfusion injury in a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of a reperfusion injury in a subject, comprisingadministering to the subject an inhibitor of an HDAC, thereby reducingan extent of a reperfusion injury in a subject.

In another embodiment, the present invention provides a method ofreducing a size of a myocardial infarction in a subject, comprisingadministering to the subject an inhibitor of an HDAC, thereby reducing asize of a myocardial infarction in a subject.

In another embodiment, the present invention provides a method ofreducing the morbidity of a myocardial infarction in a subject,comprising administering to the subject an inhibitor of an HDAC, therebyreducing the morbidity of a myocardial infarction in a subject.

In one embodiment, the HDAC inhibitor used in methods of the presentinvention is valproate. In another embodiment, the HDAC inhibitor istrichostatin. In another embodiment, the HDAC inhibitor is trichostatinA (TSA). In another embodiment, the HDAC inhibitor is Scriptaid. Inanother embodiment, the HDAC inhibitor is a PXD101.

In another embodiment, the HDAC inhibitor is a short-chain fatty acid.In another embodiment, the short-chain fatty acid is a butyrate. Inanother embodiment, the short-chain fatty acid is a phenylbutyrate. Inanother embodiment, the short-chain fatty acid is valproate. In anotherembodiment, the short-chain fatty acid is any other short-chain fattyacid that exhibits HDAC inhibitory activity. Each short-chain fatty acidrepresents a separate embodiment of the present invention.

In another embodiment, the HDAC inhibitor is a hydroxamic acid. In oneembodiment, the hydroxamic acid is a suberoylanilide hydroxamic acid(SAHA). In another embodiment, the hydroxamic acid is a derivative of aSAHA. In another embodiment, the hydroxamic acid is oxamflatin. Inanother embodiment, the hydroxamic acid is ABHA. In another embodiment,the hydroxamic acid is pyroxamide. In another embodiment, the hydroxamicacid is a propenamide. In another embodiment, the hydroxamic acid is anyother hydroxamic acid that exhibits HDAC inhibitory activity. Eachhydroxamic acid represents a separate embodiment of the presentinvention.

In another embodiment, the HDAC inhibitor is an epoxyketone-containingcyclic tetrapeptide. In one embodiment, the epoxyketone-containingcyclic tetrapeptide is a trapoxin. In another embodiment, theepoxyketone-containing cyclic tetrapeptide is an HC-toxin. In anotherembodiment, the epoxyketone-containing cyclic tetrapeptide ischlamydocin. In another embodiment, the epoxyketone-containing cyclictetrapeptide is ABHA. In another embodiment, the epoxyketone-containingcyclic tetrapeptide is pyroxamide. In another embodiment, theepoxyketone-containing cyclic tetrapeptide is a diheteropeptin. Inanother embodiment, the epoxyketone-containing cyclic tetrapeptide isWF-3161. In another embodiment, the epoxyketone-containing cyclictetrapeptide is a Cyl-2. In another embodiment, theepoxyketone-containing cyclic tetrapeptide is a Cyl-1. In anotherembodiment, the epoxyketone-containing cyclic tetrapeptide is any otherepoxyketone-containing cyclic tetrapeptide that exhibits HDAC inhibitoryactivity. Each epoxyketone-containing cyclic tetrapeptide represents aseparate embodiment of the present invention.

In another embodiment, the HDAC inhibitor is anon-epoxyketone-containing cyclic tetrapeptide. In one embodiment, thenon-epoxyketone-containing cyclic tetrapeptide is ER901228. In anotherembodiment, the non-epoxyketone-containing cyclic tetrapeptide is anapicidin. In another embodiment, the non-epoxyketone-containing cyclictetrapeptide is a cyclic-hydroxamic-acid-containing peptide (CHAP). Inanother embodiment, the non-epoxyketone-containing cyclic tetrapeptideis any other non-epoxyketone-containing cyclic tetrapeptide thatexhibits HDAC inhibitory activity. Each non-epoxyketone-containingcyclic tetrapeptide represents a separate embodiment of the presentinvention.

In another embodiment, the HDAC inhibitor is a benzamide. In oneembodiment, the benzamide is MS-275 (MS-27-275). In another embodiment,the benzamide is CI-994. In another embodiment, thenon-epoxyketone-containing cyclic tetrapeptide is any othernon-epoxyketone-containing cyclic tetrapeptide that exhibits HDACinhibitory activity. Each non-epoxyketone-containing cyclic tetrapeptiderepresents a separate embodiment of the present invention.

In another embodiment, the HDAC inhibitor is a depudecin. In anotherembodiment, the HDAC inhibitor is an organosulfur compound. In anotherembodiment, the HDAC inhibitor is any other HDAC inhibitor known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a method of present invention further comprisesadministering to the subject an inhibitor of a DNA methyltransferase(DNMT), in addition to the HDAC inhibitor. In another embodiment, anyother therapeutic compound known in the art is administered in additionto the HDAC inhibitor. In another, the HDAC inhibitor is administered asthe only active compound. Each possibility represents a separateembodiment of the present invention.

In another embodiment of methods of the present invention, the dose ofthe HDAC inhibitor is within a range of about 0.1-100 mg/day. In anotherembodiment, the dose is between about 0.5-50 mg/day. In anotherembodiment, the dose is between about 1-30 mg/day. In anotherembodiment, the dose is between about 2-20 mg/day. In anotherembodiment, the dose is between about 4-15 mg/day. In anotherembodiment, the dose is between about 6-10 mg/day.

In another embodiment, the dose is about 0.1 mg/day. In anotherembodiment, the dose is about 0.15 mg/day. In another embodiment, thedose is about 0.2 mg/day. In another embodiment, the dose is about 0.3mg/day. In another embodiment, the dose is about 0.5 mg/day. In anotherembodiment, the dose is about 1 mg/day. In another embodiment, the doseis about 1.5 mg/day. In another embodiment, the dose is about 2 mg/day.In another embodiment, the dose is about 3 mg/day. In anotherembodiment, the dose is about 5 mg/day. In another embodiment, the doseis about 7 mg/day. In another embodiment, the dose is about 10 mg/day.In another embodiment, the dose is about 15 mg/day. In anotherembodiment, the dose is about 20 mg/day. In another embodiment, the doseis about 30 mg/day. In another embodiment, the dose is about 50 mg/day.In another embodiment, the dose is about 70 mg/day. In anotherembodiment, the dose is about 100 mg/day.

In another embodiment, the dose is about 0.1 mg. In another embodiment,the dose is about 0.15 mg. In another embodiment, the dose is about 0.2mg. In another embodiment, the dose is about 0.3 mg. In anotherembodiment, the dose is about 0.5 mg. In another embodiment, the dose isabout 1 mg. In another embodiment, the dose is about 1.5 mg. In anotherembodiment, the dose is about 2 mg. In another embodiment, the dose isabout 3 mg. In another embodiment, the dose is about 5 mg. In anotherembodiment, the dose is about 7 mg. In another embodiment, the dose isabout 10 mg. In another embodiment, the dose is about 15 mg. In anotherembodiment, the dose is about 20 mg. In another embodiment, the dose isabout 30 mg. In another embodiment, the dose is about 50 mg. In anotherembodiment, the dose is about 70 mg. In another embodiment, the dose isabout 100 mg. The dose administered, the frequency of administration andthe duration of the treatment will vary, in another embodiment, as afunction of the condition of the patient and is determined according tostandard clinical procedures known to the practitioner skilled in therelevant art. Each dose or range thereof represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forameliorating an ischemic injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition fortreating an ischemic injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing an extent of an ischemic injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forameliorating an ischemia-reperfusion injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing an extent of an ischemia-reperfusion injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forameliorating a reperfusion injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing an extent of a reperfusion injury.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing a size of a myocardial infarction.

In another embodiment, the present invention provides a use of a DNMTinhibitor for the preparation of a pharmaceutical composition forreducing a morbidity of a myocardial infarction.

As provided herein, the findings in Example 1 and 3 demonstrate thatDNMT inhibition reduces myocardial infarct volume in a clinicallyrelevant model of myocardial infarction. Thus, DNMT inhibitors havemultiple applications in both treating and reducing the incidence oftissue injury associated with ischemia and subsequent reperfusion,reducing morbidity and mortality of myocardial infarction, etc.

In another embodiment, the present invention provides a method ofameliorating an ischemic injury in a tissue of a subject, comprisingadministering to the subject an inhibitor of a DNMT, therebyameliorating an ischemic injury in a tissue of a subject.

In another embodiment, the present invention provides a method oftreating an ischemic injury in a tissue of a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby treating anischemic injury in a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of an ischemic injury in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby reducing anextent of an ischemic injury in a subject.

In another embodiment, the present invention provides a method ofreducing an incidence of an ischemic injury in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby treating anischemic injury in a subject.

In another embodiment, the present invention provides a method ofameliorating an ischemia-reperfusion injury in a tissue of a subject,comprising administering to the subject an inhibitor of a DNMT, therebyameliorating an ischemia-reperfusion injury in a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of an ischemia-reperfusion injury in a subject,comprising administering to the subject an inhibitor of a DNMT, therebyreducing an extent of an ischemia-reperfusion injury in a subject.

In another embodiment, the present invention provides a method ofameliorating a reperfusion injury in a tissue of a subject, comprisingadministering to the subject an inhibitor of a DNMT, therebyameliorating a reperfusion injury in a tissue of a subject.

In another embodiment, the present invention provides a method ofreducing an extent of a reperfusion injury in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby reducing anextent of a reperfusion injury in a subject.

In another embodiment, the present invention provides a method ofreducing a size of a myocardial infarction in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby reducing asize of a myocardial infarction in a subject.

In another embodiment, the present invention provides a method ofreducing an extent of a reperfusion injury in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby reducing anextent of a reperfusion injury in a subject.

In another embodiment, the present invention provides a method ofreducing a morbidity of a myocardial infarction in a subject, comprisingadministering to the subject an inhibitor of a DNMT, thereby reducing amorbidity of a myocardial infarction in a subject.

In one embodiment, the DNMT inhibitor used in methods of the presentinvention is 5-aza-cytidine. In another embodiment, the DNMT inhibitoris 5-aza-2′-deoxycytidine. In another embodiment, the DNMT inhibitor isMG98.

In another embodiment, the DNMT inhibitor is S-adenosyl-homocysteine(SAH) or an analogue thereof. In one embodiment, the analogue isperiodate-oxidized adenosine (Liteplo, et al, Cancer Res., 4, 577-582,1986) or 3-deazaadenosine (Aarbakke, J et al, Cancer Res., 46,5469-5472, 1986; Chiang, P et al, J. Biol. Chem., 267, 4988-4991, 1992).In another embodiment, the DNMT inhibitor is a DNA-based inhibitor suchas those described in (Bigey, P et al, J. Biol. Chem., 274, 4594-4606,1999). In another embodiment, the DNMT inhibitor is an antisensenucleotide such as those described in (Ramchandani, S et al, Proc. Natl.Acad. Sci. USA, 94, 684-689, 1997; Fournel, M et al, J. Biol. Chem.,274, 24250-24256, 1999). In another embodiment, the DNMT inhibitor isany other DNMT inhibitor known in the art. Each DNMT inhibitorrepresents a separate embodiment of the present invention.

Methods of detecting and assessing the extent of ischemic injury arewell known in the art. In one embodiment, the ischemic injury or extentthereof of is determined using fluorescent microspheres (Examples).Other methods of determining ischemic injury or the extent thereof aredescribed, for example in Vuotikka P et al (Scand Cardiovasc J. 2003;37(1): 23-9), Zovein A et al, (Am J Physiol Regul Integr Comp Physiol.2004 February; 286(2): R273-82); and Liu Z et al (J Nucl Med. 2004 July;45(7): 1251-9). Each method of detecting or assessing ischemic injuryrepresents a separate embodiment of the present invention.

“Treating” a disease or disorder refers, in one embodiment, to arrestingthe development of the disease or disorder. In another embodiment,“treating” refers to reversing the development of the disease ordisorder. In another embodiment, “treating” refers to slowing thedevelopment of the disease or disorder. In another embodiment,“treating” refers to alleviating at least one symptom of the disease ordisorder. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, an additional therapeutic compound other than theDNMT inhibitor is administered to the subject as part of the method ofthe present invention. In another embodiment, the DNMT inhibitor is theonly active compound that is administered. Each possibility represents aseparate embodiment of the present invention.

In another embodiment of methods of the present invention, the dose ofthe DNMT inhibitor is between about 1-1000 mg/day. In anotherembodiment, the dose is between about 5-500 mg/day. In anotherembodiment, the dose is between about 10-300 mg/day. In anotherembodiment, the dose is between about 20-200 mg/day. In anotherembodiment, the dose is between about 40-150 mg/day. In anotherembodiment, the dose is between about 60-100 mg/day.

In another embodiment, the dose is about 1 mg/day. In anotherembodiment, the dose is about 2 mg/day. In another embodiment, the doseis about 3 mg/day. In another embodiment, the dose is about 5 mg/day. Inanother embodiment, the dose is about 10 mg/day. In another embodiment,the dose is about 15 mg/day. In another embodiment, the dose is about 20mg/day. In another embodiment, the dose is about 30 mg/day. In anotherembodiment, the dose is about 50 mg/day. In another embodiment, the doseis about 70 mg/day. In another embodiment, the dose is about 100 mg/day.In another embodiment, the dose is about 150 mg/day. In anotherembodiment, the dose is about 200 mg/day. In another embodiment, thedose is about 300 mg/day. In another embodiment, the dose is about 500mg/day. In another embodiment, the dose is about 1000 mg/day.

In another embodiment, the dose is about 1 mg. In another embodiment,the dose is about 2 mg. In another embodiment, the dose is about 3 mg.In another embodiment, the dose is about 5 mg. In another embodiment,the dose is about 10 mg. In another embodiment, the dose is about 15 mg.In another embodiment, the dose is about 20 mg. In another embodiment,the dose is about 30 mg. In another embodiment, the dose is about 50 mg.In another embodiment, the dose is about 70 mg. In another embodiment,the dose is about 100 mg. In another embodiment, the dose is about 150mg. In another embodiment, the dose is about 200 mg. In anotherembodiment, the dose is about 300 mg. In another embodiment, the dose isabout 500 mg. In another embodiment, the dose is about 1000 mg. The doseadministered, the frequency of administration and the duration of thetreatment will vary, in another embodiment, as a function of thecondition of the patient and is determined according to standardclinical procedures known to the practitioner skilled in the relevantart. Each dose or range thereof represents a separate embodiment of thepresent invention.

In one embodiment, an active compound (e.g. an HDAC inhibitor or DNMTinhibitor) of a method of the present invention is administeredsystemically. In another embodiment, the compound is administeredlocally at or near the site of the ischemia. In another embodiment, thecompound is administered in such as way as to be concentrated at thesite of ischemia. In another embodiment, the compound is administered insuch as way as to the site of ischemia by diffusion or any other activetransport or passive transport process known in the art by whichcompounds circulate within the body. In another embodiment, the compoundis administered to the ischemic tissue. Each possibility represents aseparate embodiment of the present invention.

The pharmaceutical composition containing the HDAC inhibitor or DNMTinhibitor is, in one embodiment, administered to a subject by any methodknown to a person skilled in the art, such as parenterally,paracancerally, transmucosally, transdermally, intramuscularly,intravenously, intradermally, subcutaneously, intraperitonealy,intraventricularly, intracranially, intravaginally or intratumorally.

In another embodiment, the pharmaceutical compositions are administeredorally, and thus is formulated in a form suitable for oraladministration, i.e. as a solid or a liquid preparation. Suitable solidoral formulations include, for example, tablets, capsules, pills,granules, pellets and the like. Suitable liquid oral formulationsinclude solutions, suspensions, dispersions, emulsions, oils and thelike. In one embodiment of the present invention, the HDAC inhibitor orDNMT inhibitor is formulated in a capsule. In accordance with thisembodiment, the compositions of the present invention comprises, inaddition to the HDAC inhibitor or DNMT inhibitor active compound and theinert carrier or diluent, a hard gelating capsule.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intraarterial, or intramuscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intraarterially, and are thus formulatedin a form suitable for intraarterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces, and thus are formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the HDAC inhibitor, DNMT inhibitor or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like is prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

Further, in another embodiment, the pharmaceutical compositions areadministered as a suppository, for example a rectal suppository or aurethral suppository. Further, in another embodiment, the pharmaceuticalcompositions are administered by subcutaneous implantation of a pellet.In a further embodiment, the pellet provides for controlled release ofthe HDAC inhibitor or DNMT inhibitor over a period of time.

Pharmaceutically acceptable carriers or diluents are well known to thoseskilled in the art. The carrier or diluent is, in one embodiment, asolid carrier or diluent for solid formulations, a liquid carrier ordiluent for liquid formulations, or mixtures thereof.

Solid carriers/diluents include, but are not limited to, a gum, a starch(e.g. corn starch, pregeletanized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.microcrystalline cellulose), an acrylate (e.g. polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers areaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, and glycols such as propylene glycols orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, and fish-liver oil.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating s (e.g. cornstarch, potato starch, alginic acid, silicondioxide, croscarmelose sodium, crospovidone, guar gum, sodium starchglycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pHand ionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), protease inhibitors, surfactants (e.g. sodiumlauryl sulfate), permeation enhancers, solubilizers (e.g., glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing s(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose,guar gum), sweetners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming s (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the pharmaceutical compositions provided herein arecontrolled release compositions, i.e. compositions in which the HDACinhibitor or DNMT inhibitor is released over a period of time afteradministration. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g. fatty acids, waxes, oils). Inanother embodiment, the composition is an immediate release composition,i.e. a composition in which all of the HDAC inhibitor or DNMT inhibitoris released immediately after administration.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the composition is administeredusing intravenous infusion, an implantable osmotic pump, a transdermalpatch, liposomes, or other modes of administration. In one embodiment, apump is used (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989). In another embodiment, polymeric materials canbe used. In yet another embodiment, a controlled release system can beplaced in proximity to the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984). Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990).

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art, for example by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the HDAC inhibitor or DNMT inhibitor or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are mixed with additives customary for this purpose, suchas vehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions. For parenteral administration, the HDAC inhibitor or DNMTinhibitor or their physiologically tolerated derivatives such as salts,esters, N-oxides, and the like are converted into a solution,suspension, or emulsion, if desired with the substances customary andsuitable for this purpose, for example, solubilizers or other.

In another embodiment, the active component is formulated into thecomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule), which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed from the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

In another embodiment, the salts of the HDAC inhibitor or DNMT inhibitorare pharmaceutically acceptable salts. Other salts are, in oneembodiment, useful in the preparation of the compounds according to theinvention or of their pharmaceutically acceptable salts. Suitablepharmaceutically acceptable salts of the compounds of this inventioninclude acid addition salts which may, for example, be formed by mixinga solution of the compound according to the invention with a solution ofa pharmaceutically acceptable acid such as hydrochloric acid, sulphuricacid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid,acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid.

In another embodiment, the ischemic tissue treated therapeutically orprophylactically in the present invention is cardiac tissue. In oneembodiment, the ischemic tissue is cardiac muscle tissue. In anotherembodiment, the ischemic tissue is brain tissue. In another embodiment,the ischemic tissue is lung tissue. In another embodiment, the ischemictissue is any other ischemic tissue known in the art. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, the ischemic injury or reperfusion injury that istreated therapeutically or prophylactically is a result of a myocardialinfarction. In another embodiment, the ischemic injury is a result ofcardiac surgery. In another embodiment, the ischemic injury is a resultof a stroke. In another embodiment, the ischemic injury is a result ofany other event or medical procedure that induces ischemia. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the HDAC inhibitor or DNMT inhibitor is administeredprior to the ischemia, ischemia-inducing event, or reperfusion of thetissue. In another embodiment, the HDAC inhibitor or DNMT inhibitor isadministered concomitantly with same. In another embodiment, the HDACinhibitor or DNMT inhibitor is administered after same.

In another embodiment, the HDAC inhibitor or DNMT inhibitor isadministered within 10 minutes after the ischemia, ischemia-inducingevent, or reperfusion of the tissue. In another embodiment, the intervalafter same is 20 minutes. In another embodiment, the interval is 30minutes. In another embodiment, the interval is 40 minutes. In anotherembodiment, the interval is 50 minutes. In another embodiment, theinterval is 1 hour. In another embodiment, the interval is 1.5 hours. Inanother embodiment, the interval is 2 hours. In another embodiment, theinterval is 3 hours.

Each time of administration of the HDAC inhibitor or DNMT inhibitorrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofidentifying a compound useful for a treatment or inhibition of anischemic injury or reperfusion injury to a tissue, comprising (a)contacting a first sample of the tissue or an extract thereof with thecompound; (b) determining an experimental HDAC activity level of thefirst sample or extract thereof; (c) determining a control HDAC activitylevel of a second sample of the tissue or an extract thereof, whereinthe second sample of or extract thereof has not been contacted with thecompound; and (d) comparing the experimental HDAC activity level withthe control HDAC activity level. In one embodiment of this method, areduction of the HDAC activity by the test compound indicates utility ofthe test compound for a treatment or inhibition of an ischemic injury orreperfusion injury to the tissue.

In another embodiment, the present invention provides a method ofidentifying a compound useful for a treatment or inhibition of anischemic injury or reperfusion injury to a tissue, comprising (a)contacting a first sample of the tissue or an extract thereof with thecompound; (b) determining an experimental DNMT activity level of thefirst sample or extract thereof; (c) determining a control DNMT activitylevel of a second sample of the tissue or an extract thereof, whereinthe second sample of or extract thereof has not been contacted with thecompound; and (d) comparing the experimental DNMT activity level withthe control DNMT activity level. In one embodiment of this method, areduction of the DNMT activity by the test compound indicates utility ofthe test compound for a treatment or inhibition of an ischemic injury orreperfusion injury to the tissue.

As provided herein, the assays described in Examples 4 and 6 can be usedto screen compounds for their ability to inhibit HDAC activity, and,thus, for their utility in treating and reducing the extent of ischemicinjury and reperfusion injury. The assays described in Examples 5 and 7can be used to screen compounds for their ability to inhibit DNMTactivity, and, thus, for their utility in treating and reducing theextent of ischemic injury and reperfusion injury.

HDAC inhibition assays are well known in the art, and are described,e.g. in Kang J et al (Chem Biol Interact. 2004 Jul. 20; 148(3): 115-23)and in Liu C et al (Methods Mol Biol. 2004; 287: 87-97). Additional DNMTinhibition assays are described in Kim B Y et al (Anal Biochem. 2004Mar. 1; 326(1): 21-4) or in Yan L et al (Cancer Biol Ther. 2003September-October; 2(5):552-6). Each method of assessing inhibition ofHDAC or DNMT represents a separate embodiment of the present invention.

Thus, novel compounds having utility in treating and reducing theincidence of ischemic injury can be identified by the methods of thepresent invention.

Example 1 Inhibition of DNMT Reduces Myocardial Infarct Volume Materialsand Experimental Methods Materials

5-aza-2-deoxycytidine (5-aza-2′dC) was obtained from Sigma-Aldrich.

Experimental Design

Mice were injected with 5-aza-2′dC or saline, then subjected toischemia. The left anterior descending (LAD) coronary artery wasidentified 1 mm inferior to the left atrial appendage at which point a7-0 silk suture was passed underneath the artery and tied over a 2 mmsegment of PE-10 tubing. Both visual blanching and ST segment elevationon continuous ECG display confirmed myocardial ischemia. After 45minutes, the PE-10 tubing was removed, permitting reperfusion. Thesternotomy wound was closed in two layers with 4-0 Vicryl (Ethicon,Inc., Sommerville, N.J.) and the mouse extubated. 24 hours later, themice were sacrificed, and infarcted and at-risk myocardial tissue wasquantitated to determine infarct size.

Quantitation of Infarcted Myocardial Tissue

The volume of viable myocardium remaining was determined by incubationof the tissue with 2% triphenyltetrazolium chloride: mice wereeuthanized by CO₂ inhalation, the hearts were removed, and the aortacannulated with a 21-gauge blunt-ended needle. Hearts were perfused with3 ml of phosphate buffered saline solution, followed by 3 ml of 1%(2,3,5)-triphenyltetrazolium (TTC) at 37° C. The hearts were frozen at−80° C. for 20 minutes and cut into 1.5-mm slices. Each slice wasindividually weighed and photographed with incident light on both sideswith a Leica DC200 CCD camera and MZ16 stereomicroscope (LeicaMicrosystems, Inc.) Infarct area was determined by visualizing TTCprecipitate-excluded areas using NIH Image J software.

Quantitation of at-Risk Myocardial Tissue

The volume of tissue at risk for ischemic injury (i.e. tissue lackingnormal blood flow during the occlusion) was assessed by injection of themice with FluoSphere 15-micron fluorescent microspheres (MolecularProbes/Invitrogen, Eugene, Oreg.) in the ascending aorta (clampeddistally) after re-ligation of the left anterior descending coronaryartery. The heart was sectioned, and fluorescence measured by cuttingthe tissue into 100 μm slices and determining the area of microsphereperfused cardiac tissue (perfused tissue) and the area excluded frommicrospheres (area at risk).

Statistical Analysis

The volume of necrotic tissue was divided by the volume of tissue atrisk for ischemic injury, to obtain the infarct size. Infarct size inthe drug-treated groups was compared to the infarct size in thevehicle-treated group to determine the relative reduction in injuryreported in the Table.

Results

To ascertain the effect of DNMT inhibition on ischemic injury, mice wereinjected in the left ventricle with either the DNMT inhibitor5-aza-2-deoxycytidine (5-aza-2′dC) or saline (negative control), thencardiac ischemia was induced by occlusion of the left anteriordescending coronary artery for 45 minutes. The hearts were re-perfusedfor 24 hours, and the mice were sacrificed. Inhibition of DNMT reducedinfarct size by 69.6% (33.9%/10.3%), as shown below in Table 1.

TABLE 1 5-aza-2-deoxycytidine reduces myocardial infarct size. TreatmentRatio of infarct size/area at risk Saline 33.9% 5-aza-2′dC 10.3%

The results of this Example demonstrate that inhibition of DNMT reducesinjury resulting from ischemia and reperfusion, and is useful intreating and/or reducing the incidence of same.

Example 2 Inhibition of HDAC Reduces Myocardial Infarct Volume Materialsand Experimental Methods TSA

TSA was obtained from Sigma-Aldrich.

Results

Mice were administered either the HDAC inhibitor TSA or vehicle (DMSO)alone, in the left ventricle. Ischemia was induced by occlusion of theleft anterior descending coronary artery for 45 minutes, and mice weresacrificed following a 24-hour reperfusion period. The volume ofinfarcted and at-risk myocardial tissue was quantitated as described forExample 1. As depicted below in Table 2, TSA treatment resulted in a48.3% reduction in infarct size.

TABLE 2 TSA reduces myocardial infarct size. Treatment Ratio of infarctsize/area at risk DMSO 36.0% TSA 18.6%

The results of this Example demonstrate that inhibition of HDAC reducesinjury resulting from ischemia and reperfusion, and is useful intreating and/or reducing the incidence of same.

Example 3 HDAC Inhibitors and DNMT Inhibitors Reduce Ischemic Injurywhen Administered after Onset of Ischemia

To further characterize the effect of HDAC inhibitors and DNMTinhibitors on ischemic injury, a larger study was conducted, in whichthe HDAC inhibitor was added at several time points. In addition, adifferent HDAC inhibitor, Scriptaid (BioMol, Plymouth Meeting, Pa.), wastested, together with its corresponding negative control compound,Nullscript (Biomol). Ischemia was induced, and amounts of viable andnecrotic tissue measured, as described for Example 1. A total of 217mice were utilized in this Example, distributed as follows: Saline=38;DMSO=45; Nullscript=11; TSA-T1=31; TSA-T2=25; TSA-T3=15; Scriptaid=25;5aza2′dC=27. As depicted in Table 3, both HDAC inhibitors significantlyreduced infarct size. In addition, HDAC inhibitors were effective whenadministered either before ischemia or 1 hour after ischemia.

The results presented in this Example confirm the results of theprevious Example, and show in addition that the effect of HDACinhibitors and DNMT inhibitors is not limited to a particular compound,but rather various HDAC inhibitors and DNMT inhibitors can be utilized.In addition, the results show that HDAC inhibitors and DNMT inhibitorscan reduce ischemia-reperfusion injury when added either before or afterthe onset of ischemia.

TABLE 3 Effects of HDAC inhibitors and DNMT inhibitors onischemia-reperfusion injury. Infarct size/ p value % decrease area atrisk (w.r.t. (w.r.t. Class Treatment (±SEM) DMSO) DMSO) Control DMSO36.0% ± 0.04% — — Nullscript 38.0% ± 0.56% — — HDAC TSA (T1) 18.6% ±0.04% p = 0.015 48.30% inhibitor TSA (T2) 16.0% ± 0.04% p = 0.013 55.60%TSA (T3) 33.0% ± 0.05% p = 0.674  8.30% Scriptaid (T1) 21.2% ± 0.33% p =0.035 44.20% DNMT 5-aza-2′dC 10.3% ± 0.03% p = 0.004 71.40% inhibitor(T1—treatment 1 hour before ischemia; T2—treatment 1 hour afterischemia; T3—treatment 12 hours after ischemia)

Example 4 Global Histone H3 Acetylation is Down-Regulated DuringIschemia; this Reduction is Prevented by HDAC Inhibitors Materials andExperimental Methods Harvesting of Cells and Histone Isolation

Cells were lysed in Triton X/deoxycholate buffer (20 millimolar [mM]HEPES, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 100 mM sodiumchloride, 50 mM sodium fluoride, 5 mM EDTA, 100 μM sodium molybdate,with protease inhibitors), and insoluble material was removed by a 15minute (min) centrifugation in a microfuge, and the supernatant wassubjected to 100,000×g, 30 mM centrifugation. The pellet was extractedwith 9 M urea, and the resulting pellet extracted with 0.3 M HCl.

Electrophoresis and Western Blotting

40 centimeter (cm) acetic acid/urea gels were prepared as follows: Theseparating gel (0.75 millimeter [mm] thick) contained 15%acrylamide/0.2% methylenebisacrylamide, 4 molar (M) urea, and 5% aceticacid. The stacking gel contained 7.5% acrylamide/0.1%methylenebisacrylamide, 8 M urea, and 5% acetic acid. Proteins weresolubilized in sample buffer (8 M urea, 5% acetic acid, 5%2-mercaptoethanol, 25 milligram per milliliter (mg/ml) protaminesulfate, with methyl green) and loaded onto gels that had beenpre-electrophoresed to a constant current and scavenged with 2.5 Mcysteamine (Sigma-Aldrich).

After electrophoresis, the gels were transferred to PVDF membranes using0.1% acetic acid/10% methanol as a transfer buffer, and Western blottedwith anti-acetyl H3 (Upstate, Charlottesville, Va.), which recognizesacetyl-Lys9 H3 peptides.

Results

To determine the effect of ischemia and HDAC inhibitors on histoneacetylation, ischemia was induced in mice, as described in Example 1,and the degree of histone acetylation in myocardium was determinedHistone acetylation was reduced in response to ischemia (FIG. 1).Pre-treatment with T1, and, to a lesser extent, administration of T1 1hour after the onset of ischemia, partially prevented the reduction inhistone acetylation.

These findings confirm the results of the previous Examples. Examples1-4 show that alteration of chromatin structure in response to ischemiacauses ischemia-reperfusion injury, and that by preventing or inhibitingthis alteration, using HDAC or DNMT inhibitors, ischemia-reperfusioninjury can be significantly reduced.

Example 5 Testing of Compounds for Inhibition of HDAC Activity inCardiac Myocyte Extracts

HDAC activity of cardiac myocyte extracts is assessed as described inExample 4, in the presence and absence of a test compound.

This assay can be used to identify novel agents to treat and reduceischemic injury, as inhibition of HDAC activity is in the presentinvention to reduce ischemic injury.

Example 6 Testing of Compounds for Inhibition of DNMT Activity in aCellular DNMT Assay Materials and Experimental Methods Cardiac MyocyteCultures

Cardiac myocyte cultures are generated and maintained as described in(Sherry B et al, J Virol 70: 6709-15, 1996).

DNMT Assay

DNMT activity is assessed by measuring the methylation state of cellularDNA by incubation with 4 U of SssI CpG methylase (New England Biolabs)in the presence of 1.5 μM S-adenosyl-L-[methyl-3^(H)]methionine, asdescribed in (Fang J et al, J Virol, 75(20): 9753-9761, 2001).

Results

DNMT activity is assessed in the presence and absence of a testcompound. This assay can be used to identify novel agents to treat andreduce ischemic injury, as inhibition of DNMT activity is in the presentinvention to reduce ischemic injury.

1-23. (canceled)
 24. A method for reducing the size of a myocardialinfarct in a subject in need thereof, the method comprising:administering acutely to said subject a therapeutically effective amountof a histone deacetylase (HDAC) inhibitor, wherein said HDAC inhibitoris suberoylanilide hydroxamic acid (SAHA), thereby reducing the size ofsaid myocardial infarct in said subject.
 25. The method of claim 24,wherein said HDAC inhibitor is administered to said subject before orduring a cardiac surgery.
 26. The method of claim 24, wherein said HDACinhibitor is administered to said subject within one hour after acardiac surgery.
 27. The method of claim 24, wherein said HDAC inhibitoris administered to said subject within two hours after a cardiacsurgery.
 28. The method of claim 24, wherein said HDAC inhibitor isadministered to said subject within three hours after a cardiac surgery.29. The method of claim 24, wherein said HDAC inhibitor is administeredto said subject within one hour after a stroke.
 30. The method of claim24, wherein said HDAC inhibitor is administered to said subject withinone hour after a myocardial infarction.
 31. The method of claim 24,wherein said HDAC inhibitor is administered to said subject within twohours after a myocardial infarction.
 32. The method of claim 24, whereinsaid HDAC inhibitor is administered to said subject within three hoursafter a myocardial infarction.
 33. A method for treating an ischemicinjury in a subject in need thereof, the method comprising:administering acutely to said subject with said ischemic injury atherapeutically effective amount of a histone deacetylase (HDAC)inhibitor, wherein said HDAC inhibitor is suberoylanilide hydroxamicacid (SAHA), thereby treating said ischemic injury in said subject. 34.The method of claim 33, wherein said HDAC inhibitor is administered tosaid subject before or during a cardiac surgery.
 35. The method of claim33, wherein said HDAC inhibitor is administered to said subject withinone hour after a cardiac surgery.
 36. The method of claim 33, whereinsaid HDAC inhibitor is administered to said subject within two hoursafter a cardiac surgery.
 37. The method of claim 33, wherein said HDACinhibitor is administered to said subject within three hours after acardiac surgery.
 38. The method of claim 33, wherein said HDAC inhibitoris administered to said subject within one hour after a stroke.
 39. Themethod of claim 33, wherein said HDAC inhibitor is administered to saidsubject within one hour after a myocardial infarction.
 40. The method ofclaim 24, wherein said HDAC inhibitor is administered to said subjectwithin two hours after a myocardial infarction.
 41. The method of claim24, wherein said HDAC inhibitor is administered to said subject withinthree hours after a myocardial infarction.
 42. method of claim 33,wherein said ischemic injury is in a cardiac tissue.